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In this study, waxy corn starch was modified with 230 U or 460 U of amylosucrase (AS) from Neisseria polysaccharea (NP) to elongate the glucan. The amylose content of the AS-modified starches was determined using iodine and concanavalin A (Con A) methods, and their in vivo digestion, thermal, swelling, and pasting properties were evaluated. The amylose content of AS-treated starches was not significantly different (p > 0.05) when using the Con A method but was significantly higher than that of non-AS-treated samples when using the iodine method. In vivo, rats fed AS-treated starch had significantly lower blood glucose levels at 15 min than other rats; rats fed 460 U AS had lower blood glucose levels at 30 and 60 min than non-AS-treated rats. DSC analysis revealed that AS-treated starches exhibited higher initial, melting, and completion temperatures. Minimal volume expansion was observed by swelling factor analysis, while a Rapid Visco Analyzer assessment revealed that they had higher pasting onset temperatures, lower peak viscosities, and no trough viscosity compared to native starch. The elongated glucans in AS-treated starch reinforced their crystalline structure and increased slowly digestible and enzyme-resistant starch content. Overall, AS-treated starch showed unique thermal properties and a reduced blood glucose index upon administration. This distinctive characteristic of NPAS-treated starch makes it a good candidate food or non-food material for cosmetic products, medical materials, and adhesives. [ABSTRACT FROM AUTHOR]

Our pursuit of new compounds with enhanced bioavailability and bioactivity prompted us to employ the biotransformation-guided purification (BGP) approach which leverages proficient in vitro biotransformation techniques. Angelica dahurica roots, also called Baizhi in Chinese traditional medicine, are famous for their anti-inflammatory and analgesic properties. Herein, we applied the BGP methodology to Baizhi extracts, employing Deinococcus geothermalis amylosucrase (Dg AS), an enzyme demonstrating catalytic competence across diverse substrates, for biotransformation. Initiating with a 70 % methanol extraction, we obtained the crude extract of commercial Baizhi powder, followed by an additional extraction using ethyl acetate. Notably, reactions performed on this extract yielded limited quantities of novel compounds. Subsequently, the extract underwent partitioning into four fractions based on HPLC profiling, leading to the successful isolation of a compound with significant yield from fraction 2 mixtures upon reaction with Dg AS. Structural elucidation confirmed the compound as byakangelicin-7″- O - α -glucopyranoside (BG-G), a new alpha glycoside derivative of byakangelicin. Furthermore, validation experiments verified the capacity of Dg AS to glycosylate pure byakangelicin, yielding BG-G. Remarkably, the aqueous solubility of BG-G exceeded that of byakangelicin by over 29,000-fold. In conclusion, BGP emerges as a potent strategy combining traditional medicinal insights with robust enzymatic tools for generating new compounds. [ABSTRACT FROM AUTHOR]

Naringenin, a flavanone (flavonoid family) has bioactive effects, such as antioxidant, free radical scavenging, anti-inflammatory, carbohydrate metabolism, and immunoregulatory effects on the human body. However, its low water solubility and bioavailability limit its use in food, pharmaceutical, cosmetic, and drug industries. Amylosucrase (ASase, E. C. 2.4.1.4), uses sucrose as a substrate and is a glycosyltransferase that hydrolyzes glucose and fructose generating a glycosylated product. In this study, the putative ASase gene (accession number: WP_102128133.1) from Deinococcus planocerae KCTC 33809 (DplAS) was expressed in Escherichia coli. The optimum reaction temperature and pH for sucrose hydrolytic capacity of DplAS were determined to be 30 ℃ and 7.0, respectively. DplAS was determined to have a half-life of 256.7 and 42.8 min at 40 ℃ and 45 ℃, respectively. Although it exhibited high similarity with DgAS (80%), DplAS displayed different acceptor specificities to that of DgAS. Uniquely, DplAS generated one major product (naringenin-maltoside) via transglycosylation using sucrose and naringenin as donor and acceptor molecules, respectively. Based on NMR, naringenin-maltoside was identified as naringenin-4'-O-a-D-maltopyranoside, and its conversion rate was optimized using the response surface method (RSM) with central composite design (CCD). This resulted in a donor to acceptor ratio of 25.9:1 at 27.64 ℃, and a 55.69% conversion of sucrose. [ABSTRACT FROM AUTHOR]

Narrow fractions of short-chain enzymatically-synthesized amylose have been crystallized from 0.05 to 5.0% w/v aqueous solutions by slow diffusion of acetone or ethanol vapors at 50–60 °C or addition of hot liquid acetone. The fractions were selected according to their ability to form a few micrometer-long A-type acicular single crystals with a well-defined facetted habit. The single crystals grew organized into rosettes or fan-like assemblies whose relative fraction depended on the initial amylose concentration. Rosettes became dominant with increasing concentration while the aspect ratio of the constituting crystals increased with crystallization temperature. In rosettes, the density of crystals increased as well but without forming solid spherulites, due to the topological constraint of packing isomorphous platelet crystals growing from a common nucleus inside a spherical volume. A higher density of crystals was achieved by nucleation on the surface defects of inductive glass slides. Due to the chemical polarity of A-type amylose single crystals, rosettes and crystal mats exposed faces concentrating reducing amylose chain ends, available for chemical labelling or grafting. [ABSTRACT FROM AUTHOR]

A biocatalytic system that could produce bioactive resveratrol poly-glucosides, using sucrose as a low-cost source of UDP-glucose donors and amylosucrase DgAS from Deinococcus geothermalis, was developed in this study. This system boasts several advantages, including the rapid and direct conversion of substrates to products, thermostability, regio-stereospecificity, and effectiveness, both in vitro and in vivo, at 40 °C. The results showed that the optimal reaction condition of the production of resveratrol glucosides was obtained by 2.0 µg/mL DgAS and 100 mM sucrose at pH 7.0, incubated at 40 °C for 5 h. With a success rate of around 97.0% in vitro and 95.0% in vivo in a short period of time, resveratrol-O-glucosides showed exciting outcomes in cosmetic applications, including antioxidant, anti-inflammatory, anti-aging, and whitening effects when tested with Raw 264.7, B16, and HS68 cell lines. DgAS is recognized as an important biocatalyst due to its high thermostability, effectiveness, and specificity among all known amylosucrases (ASases) in the production of poly-glucosides in a chain of polyphenols, such as resveratrol, making it an ideal candidate for industrial use in the cost-effective production of cosmetic items. [ABSTRACT FROM AUTHOR]

In this study, waxy corn starch was modified with 230 U or 460 U of amylosucrase (AS) from Neisseria polysaccharea (NP) to elongate the glucan. The amylose content of the AS-modified starches was determined using iodine and concanavalin A (Con A) methods, and their in vivo digestion, thermal, swelling, and pasting properties were evaluated. The amylose content of AS-treated starches was not significantly different (p > 0.05) when using the Con A method but was significantly higher than that of non-AS-treated samples when using the iodine method. In vivo, rats fed AS-treated starch had significantly lower blood glucose levels at 15 min than other rats; rats fed 460 U AS had lower blood glucose levels at 30 and 60 min than non-AS-treated rats. DSC analysis revealed that AS-treated starches exhibited higher initial, melting, and completion temperatures. Minimal volume expansion was observed by swelling factor analysis, while a Rapid Visco Analyzer assessment revealed that they had higher pasting onset temperatures, lower peak viscosities, and no trough viscosity compared to native starch. The elongated glucans in AS-treated starch reinforced their crystalline structure and increased slowly digestible and enzyme-resistant starch content. Overall, AS-treated starch showed unique thermal properties and a reduced blood glucose index upon administration. This distinctive characteristic of NPAS-treated starch makes it a good candidate food or non-food material for cosmetic products, medical materials, and adhesives.

Amylosucrase can increase the amount of resistant starch (RS) in starch by transferring glucose from sucrose to amylopectin. Here, rice starch was modified using amylosucrase from Deinococcus geothermalis (DgAS). DgAS-modified rice starch (DMRS) increased the side-chain length of amylopectin and appeared in the form of B-type crystals. In vitro digestion analyses revealed that DMRS had a higher RS contents and lower digestion rate than native rice starch. When high-fat diet (HFD)-induced C57BL/6 mice were orally administered DMRS, body weight and white fat tissues of DMRS-fed HFD mice were not significantly different. However, serum leptin and glucose levels were significantly decreased and serum glucagon like peptide-1was increased in these mice. The cecal microbiome in DMRS-fed HFD mice was identified to investigate the role of DMRS in gut microbiota regulation. DMRS supplementation increased the relative abundance of Bacteroides, Faecalibaculum, and Ruminococcus in mouse gut microbiota. [ABSTRACT FROM AUTHOR]

In this study, a novel high-activity amylosucrase from Salinispirillum sp. LH10-3-1 (SaAS) was identified and characterized. The recombinant enzyme was determined as a monomer with a molecular mass of 75 kDa. SaAS protein exhibited the maximum total and polymerization activities at pH 9.0 and maximum hydrolysis activity at pH 8.0. The optimum temperature for total, polymerization, and hydrolysis activities were 40, 40, and 45 °C, respectively. Under the optimal pH and temperature, SaAS had a specific activity of 108.2 U/mg. SaAS also showed excellent salt tolerance and could retain 77.4% of its original total activity at 4.0 M NaCl. The addition of Mg2+, Ba2+, and Ca2+ enhanced the total activity of SaAS. When the conversion of 0.1 M and 1.0 M sucrose was catalyzed at pH 9.0 and 40 °C for 24 h, the ratios of hydrolysis, polymerization, and isomerization reactions were 11.9:77.4:10.7 and 15.3:53.5:31.2, respectively. The α-arbutin yield of 60.3% was achieved from 20 mM sucrose and 5 mM hydroquinone catalyzed by SaAS. Key points: • A novel amylosucrase from Salinispirillum sp. LH10-3-1 (SaAS) was characterized. • SaAS has the highest specific enzyme activity among all known amylosucrase. • SaAS has hydrolysis, polymerization, isomerization, and glucosyltransferase activities. [ABSTRACT FROM AUTHOR]

Amylosucrase (ASase) from Deinococcus geothermalis (DgAS) is involved in the highest number of glycotransfer reactions with different flavonoids as acceptor molecules among all ASases, overcoming the flavonoid disadvantage; however, its transglycosylation yield is low if the structure of the acceptor molecule does not fit into its active site pocket. Therefore, this study focused on improving the transglycosylation yield and elucidating the underlying mechanism of action by replacing the isoleucine at position 221 of DgAS (I221V) near subsite +1 with a valine residue. Molecular dynamics simulations revealed that the I221V mutation in the protein did not significantly alter its overall conformation but led to decreased flexibility around residues 3 and 7 and an increase in the active site pocket volume. The proportion of hydrolysis and polymerization reactions in I221V increased when reacted with sucrose as the sole substrate, while the proportion of isomerization reactions decreased compared to those of the DgAS wild-type (WT). In addition, I221V showed a higher transglycosylation efficiency for 6- and 4′-hydroxyflavones than for WT, which was also twofold higher for biosynthesized naringenin α-glucoside. Kinetic parameters showed that the higher transglycosylation efficiency of I221V compared to that of WT was due to its increased catalytic rate for sucrose as a donor molecule, rather than its affinity for naringenin as an acceptor molecule. Overall, our findings provide further insight into how DgAS mutation can affect the transglycosylation reaction, which could aid in the development of more efficient industrial applications. [ABSTRACT FROM AUTHOR]

Amylosucrase (ASase) efficiently biosynthesizes α-glucoside using flavonoids as acceptor molecules and sucrose as a donor molecule. Here, ASase from Deinococcus wulumuqiensis (DwAS) biosynthesized more naringenin α-glucoside (NαG) with sucrose and naringenin as donor and acceptor molecules, respectively, than other ASases from Deinococcus sp. The biotransformation rate of DwAS to NαG was 21.3% compared to 7.1–16.2% for other ASases. Docking simulations showed that the active site of DwAS was more accessible to naringenin than those of others. The 217th valine in DwAS corresponded to the 221st isoleucine in Deinococcus geothermalis AS (DgAS), and the isoleucine possibly prevented naringenin from accessing the active site. The DwAS-V217I mutant had a significantly lower biosynthetic rate of NαG than DwAS. The k cat /K m value of DwAS with naringenin as the donor was significantly higher than that of DgAS and DwAS-V217I. In addition, NαG inhibited human intestinal α-glucosidase more efficiently than naringenin. [Display omitted] • Naringenin α-glucoside is efficiently synthesized by D. wulumuquiensis amylosucrase. • Biosynthesized naringenin α-glucoside can be used for treating chronic diseases. • Naringenin α-glucoside might be a better functional food ingredient than naringenin. [ABSTRACT FROM AUTHOR]

The aim of this study was to establish an efficient bioprocess for the synthesis of trehalulose as a novel sweetener. This disaccharide has 70% of the sweetness of sucrose and bioactive properties such as anti-cariogenicity and anti-oxidizing activity. In this study, amylosucrase from the Deinococcus deserti (Dd AS) gene was expressed and purified. When Dd AS was reacted with 2 M sucrose at 35 °C for 120 h, the yield ratio of trehalulose to turanose was approximately 2:1. The trehalulose yield increased when extrinsic fructose was added. Under optimum conditions for trehalulose synthesis, the yield reached 36% (246 g/L, sucrose basis) starting with 2 M sucrose + 0.75 M fructose and showed the highest trehalulose productivity (1.94 g/L/h). As a result, a novel amylosucrase that synthesized trehalulose as the major product was developed, in contrast to other studied amylosucrase-type enzymes. Dd AS could be utilized industrially in a bioprocess for producing trehalulose as a functional sucrose alternative. • A novel amylosucrase was found to synthesize trehalulose as a main product. • The enzyme from Deinococcus sp. was expressed in E. coli. • Yield ratio of trehalulose to turanose was 2:1 with 2 M sucrose at 35 °C and pH7.5. • Maximum trehalulose yield reached 36% with 2 M sucrose + 0.75 M fructose. • Industrial application of this enzyme may lead to development of a valuable sweetener. [ABSTRACT FROM AUTHOR]

Amylosucrase from Neisseria polysaccharea (NpAS) synthesizes α-1,4 glucan polymer from sucrose. In this study, we coated various botanical sources of raw starch with an α-glucan layer generated by NpAS to improve physicochemical properties. Field-emission scanning electron microscopy demonstrated that all surfaces of the starch granules were successfully coated via the NpAS reaction. X-ray diffraction analysis revealed that the crystallinity decreased and the crystal pattern changed to C-type as an amylose layer formed around the surface of the starch granules. Based on rapid viscosity and differential scanning calorimetry analyses, the gelatinization resistance of the α-glucan-coated starch increased owing to decreased viscosity and increased melting temperature. Therefore, the α-glucans coated the starches by enzymatic reactions of various botanical sources; these have applicability in the food and starch industries owing to various physicochemical properties such as enhanced thermostability. [ABSTRACT FROM AUTHOR]

A biocatalytic system that could produce bioactive resveratrol poly-glucosides, using sucrose as a low-cost source of UDP-glucose donors and amylosucrase DgAS from Deinococcus geothermalis, was developed in this study. This system boasts several advantages, including the rapid and direct conversion of substrates to products, thermostability, regio-stereospecificity, and effectiveness, both in vitro and in vivo, at 40 °C. The results showed that the optimal reaction condition of the production of resveratrol glucosides was obtained by 2.0 µg/mL DgAS and 100 mM sucrose at pH 7.0, incubated at 40 °C for 5 h. With a success rate of around 97.0% in vitro and 95.0% in vivo in a short period of time, resveratrol-O-glucosides showed exciting outcomes in cosmetic applications, including antioxidant, anti-inflammatory, anti-aging, and whitening effects when tested with Raw 264.7, B16, and HS68 cell lines. DgAS is recognized as an important biocatalyst due to its high thermostability, effectiveness, and specificity among all known amylosucrases (ASases) in the production of poly-glucosides in a chain of polyphenols, such as resveratrol, making it an ideal candidate for industrial use in the cost-effective production of cosmetic items.

Puerarin (daidzein-8-C-glucoside) is an isoflavone isolated from several leguminous plants of the genus Pueraria. Puerarin possesses several pharmacological properties; however, the poor solubility of puerarin limits its applications. To resolve this poor solubility, Deinococcus geothermalis amylosucrase (DgAS) was used to modify puerarin into more soluble derivatives. The results showed that DgAS could biotransform puerarin into a novel compound: puerarin-4′-O-α-glucoside. The biotransformation reaction was manipulated at different temperatures, pH values, sucrose concentrations, reaction times, and enzyme concentrations. The results showed that the optimal reaction condition was biotransformed by 200 μg/mL DgAS with 20% (w/v) sucrose at pH 6 and incubated at 40 °C for 48 h, and the optimal production yield was 35.1%. Puerarin-4′-O-α-glucoside showed 129-fold higher solubility than that of puerarin and, thus, could be further applied for pharmacological use in the future. [ABSTRACT FROM AUTHOR]

Simple Summary: All livings are composed of cells, which contain lipid, proteins, nuclei acids, and saccharides. Saccharides include polysaccharides, oligo saccharides, disaccharides, which are linked by monosaccharides. Monosaccharides such as glucose exist in two forms, named α and β anomer, in solution. In addition, monosaccharides could be linked with lipid, proteins, nuclei acids or other saccharide to form glycosides through glycosylation. In nature, glycosylation is catalyzed by enzymes. Until now, all enzymes catalyzed glycosylation to form glycosides with either α or β form but not both. This study found an enzyme, amylosucrase from Deinococcus geothermalis (DgAS), could catalyze glycosylation of a kind of lipid named ganoderic acids triterpenoids from a medicinal fungus Ganoderma lucidum to form both α and β anomer of glycosides. This is the first report that enzymes could catalyze such glycosylation and a possible reaction mechanism was proposed. Glycosylation occurring at either lipids, proteins, or sugars plays important roles in many biological systems. In nature, enzymatic glycosylation is the formation of a glycosidic bond between the anomeric carbon of the donor sugar and the functional group of the sugar acceptor. This study found novel glycoside anomers without an anomeric carbon linkage of the sugar donor. A glycoside hydrolase (GH) enzyme, amylosucrase from Deinococcus geothermalis (DgAS), was evaluated to glycosylate ganoderic acid F (GAF), a lanostane triterpenoid from medicinal fungus Ganoderma lucidum, at different pH levels. The results showed that GAF was glycosylated by DgAS at acidic conditions pH 5 and pH 6, whereas the activity dramatically decreased to be undetectable at pH 7 or pH 8. The biotransformation product was purified by preparative high-performance liquid chromatography and identified as unusual α-glucosyl-(2→26)-GAF and β-glucosyl-(2→26)-GAF anomers by mass and nucleic magnetic resonance (NMR) spectroscopy. We further used DgAS to catalyze another six triterpenoids. Under the acidic conditions, two of six compounds, ganoderic acid A (GAA) and ganoderic acid G (GAG), could be converted to α–glucosyl-(2→26)-GAA and β–glucosyl-(2→26)-GAA anomers and α-glucosyl-(2→26)-GAG and β-glucosyl-(2→26)-GAG anomers, respectively. The glycosylation of triterpenoid aglycones was first confirmed to be converted via a GH enzyme, DgAS. The novel enzymatic glycosylation-formed glycoside anomers opens a new bioreaction in the pharmaceutical industry and in the biotechnology sector. [ABSTRACT FROM AUTHOR]

4- α -glucanotransferase is used to produce thermoreversible starch gels to alleviate limitations to use of starch gels in repetitively heat-processed foods. However, the gel strength was weakened after this enzyme modification. In the present study, treatment by amylosucrase (NpAS) of corn starch and sucrose was applied to retain the gel thermoreversibility and eliminate the shortcoming caused by 4- α -glucanotransferase (Tu α GT). Changes in molecular structure, rheological and retrogradation properties of modified starch were investigated after NpAS and Tu α GT sequential and one-pot treatment, respectively. The apparent amylose content was reduced and increased by sequential and one-pot treatments, respectively, compared to single Tu α GT modification. Chain length profiles showed higher proportion of degree of polymerization (DP) ≥ 13 by sequential treatment, whereas DP 6–12 was higher after one-pot treatment. All modified starches had reduced molecular weight. G' and G" increased by dual enzyme compared to single Tu α GT treatment having little effect on retrogradation. Interestingly, starch subjected to 3 h one-pot treatment caused G' and G" temperature curves to cross-over, improving thermoreversible properties. The results indicate that NpAS treatment compensated for loss of starch gel strength caused by Tu α GT and offered possibility to provide a wider range of thermoreversible starches. [ABSTRACT FROM AUTHOR]

Enzymatically modified isoquercitrin (EMIQ) is a glyco-chemically modified flavonoid exhibiting notably high biological activity, such as antioxidant, anti-inflammatory and anti-allergic properties. However, the utilization of expensive substrates such as isoquercitrin and cyclodextrin in the conventional approach has hindered the industrial-scale production of EMIQ due to high cost and low yields. Hence, the development of a cost-effective and efficient method is crucial for the biological synthesis of EMIQ. In this study, a natural cascade catalytic reaction system was constructed with α -L-rhamnosidase and amylosucrase using the inexpensive substrates rutin and sucrose. Additionally, a novel approach integrating gradient temperature regulation into biological cascade reactions was implemented. Under the optimal conditions, the rutin conversion reached a remarkable 95.39% at 24 h. Meanwhile, the productivity of quercetin-3- O -tetraglucoside with the best bioavailability reached an impressive 41.69%. This study presents promising prospects for future mass production of EMIQ directly prepared from rutin and sucrose. [Display omitted] • A novel cascade strategy was constructed to produce EMIQ from inexpensive substrates. • Amylosucrase with sucrose as a substrate was used to further reduce the production cost. • Gradient temperature control was innovatively integrated into the biological cascade reaction. • The conversion rate of IQ-G3 with the best bioavailability reached an impressive 41.69%. • The industrial production of EMIQ with rutin and sucrose as materials has good potential. [ABSTRACT FROM AUTHOR]

Although myricetin (3,3′,4′,5,5′,7-hexahydroxyflavone, MYR) has a high antioxidant capacity and health functions, its use as a functional food material is limited owing to its low stability and water solubility. Amylosucrase (ASase) is capable of biosynthesizing flavonol α-glycoside using flavonols as acceptor molecules and sucrose as a donor molecule. Here, ASase from Deinococcus deserti (Dd AS) efficiently biosynthesizes a novel MYR α-triglucoside (MYRαG3) using MYR as the acceptor molecule. Comparative homology analysis and computational simulation revealed that Dd AS has a different active pocket for the transglycosylation reaction. Dd AS produced MYRαG3 with a conversion efficiency of 67.4 % using 10 mM MYR and 50 mM sucrose as acceptor and donor molecules, respectively. The structure of MYRαG3 was identified as MYR 4′- O -4″,6″-tri- O -α-D-glucopyranoside using NMR and LC-MS. In silico analysis confirmed that Dd AS has a distinct active pocket compared to other ASases. In addition, molecular docking simulations predicted the synthetic sequence of MYRαG3. Furthermore, MYRαG3 showed a similar DPPH radical scavenging activity of 49 %, comparable to MYR, but with significantly higher water solubility, which increased from 0.03 μg/mL to 511.5 mg/mL. In conclusion, this study demonstrated the efficient biosynthesis of a novel MYRαG3 using Dd AS and highlighted the potential of MYRαG3 as a functional material. • Distinct active site pocket in Deinococcus deserti amylosucrase (Dd AS) compared with another ASase • Efficient biosynthesis of novel myricetin (MYR) 4′-O-4″,6″-tri-O-α-D-glucopyranoside (MYRαG3) by Dd AS • MYRαG3 showed similar antioxidant capacity and much higher water solubility than MYR. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the impacts of enzymatically synthesized α-glucans possessing α-1,4- and α-1,6-glucose linkages, and varying in branching ratio, on colonic microbiota composition and metabolic function. Four different α-glucans varying in branching ratio were synthesized by amylosucrase from Neisseria polysaccharea and glycogen branching enzyme from Rhodothermus obamensis. The branching ratios were found to range from 0 % to 2.8 % using GC/MS. In vitro fecal fermentation analyses (n = 8) revealed that the branching ratio dictates the short-chain fatty acid (SCFA) generation by fecal microbiota. Specifically, slightly branched (0.49 %) α-glucan resulted in generation of significantly (P < 0.05) higher amounts of propionate, compared to more-branched counterparts. In addition, the amount of butyrate generated from this α-glucan was statistically (P > 0.05) indistinguishable than those observed in resistant starches. 16S rRNA sequencing revealed that enzymatically synthesized α-glucans stimulated Lachnospiraceae and Ruminococcus related OTUs. Overall, the results demonstrated metabolic function of colonic microbiota can be manipulated by altering the branching ratio of enzymatically synthesized α-glucans, providing insights into specific structure-function relationships between dietary fibers and the colonic microbiome. Furthermore, the slightly branched α-glucans could be used as functional carbohydrates to stimulate the beneficial microbiota and SCFAs in the colon. [Display omitted] [ABSTRACT FROM AUTHOR]

The amylosucrase (ASase, EC 2.4.1.4) utilizes sucrose as the sole substrate to catalyze multifunctional reactions. It can naturally synthesize α-1,4-linked glucans such as amylose as well as sucrose isomers with more favorable properties than sucrose with a lower intestinal digestibility and non-cariogenic properties. The amino acid sequence of the asase gene from Deinococcus cellulosilyticus (DceAS) exhibits low homology with those of other ASases from other Deinococcus species. In this study, we cloned and expressed DceAS and demonstrated its high activity at pH 6 and pH 8 and maintained stability. It showed higher polymerization activity at pH 6 than at pH 8, but similar isomerization activity and produced more turanose and trehalulose at pH 6 than at pH 8 and produced more isomaltulose at pH 8. Furthermore, the molecular weight of DceAS was 226.6 kDa at pH 6 and 145.5 kDa at pH 8, indicating that it existed as a trimer and dimer, respectively under those conditions. Additionally, circular dichroism spectra showed that the DceAS secondary structure was different at pH 6 and pH 8. These differences in reaction products at different pHs can be harnessed to naturally produce sucrose alternatives that are more beneficial to human health. • Evaluation of pH-dependent properties of amylosucrase from D. cellulosilyticus. • Understanding differences in product formation by DceAS at pH 6 and 8 • High isomerization activity of DceAS in 50 mM sodium citrate buffer at pH 6 • Increased trehalulose/insoluble glucan ratio in 50 mM sodium citrate buffer at pH 6 [ABSTRACT FROM AUTHOR]

Glycogen-like particles (GLPs) were built up from sucrose by applying de novo one-pot enzymatic process of amylosucrase (ASase; 6 U·mL−1) and glycogen branching enzymes (GBEs; 0.001 and 0.005 U·mL−1). Due to different chain-length transferring patterns of GBEs, structurally differentiated GLPs were synthesized. Yields of GLPs synthesized at pH 7.0 and 30 °C were improved by increasing the GBE/ASase ratio. Branching degrees of GLPs obviously was increased along with the ratio of GBEs, of which result was directly supported by shortened branch-chain length with greater GBE activity. Long branch chains seemed to play as efficient acceptor molecules to bind newly transferred branch chains especially at lower ratio of GBE/ASase, resulting in greater molecular weight and size of GLP with higher proportion of them. Molecular weight, size, and density of GLPs were ranged from 7.37 × 105 to 1.94 × 108 g·mol−1, from 23.70 to 52.65 nm, and from 7.99 to 374.32 g·mol−1·nm−3, respectively. By increasing GBE/ASase ratio, more compact GLP architecture was fabricated due to increased weight and reduced size with exception of a unique GBE. GLPs were efficiently synthesized by two different glycosyltransferases, and their chemical structures were controllable by source and ratio of GBEs due to their different branch-chain transferring specificity. • Glycogen-like particle (GLP) was synthesized by one-pot dual enzyme system. • Nano-size GLP structure was highly dependent on type of branching enzymes. • Size and density of GLP were controllable by changing enzyme activity ratio. • GLP yield reached max. 44.5% by branching enzyme activity of Synechocystis sp. [ABSTRACT FROM AUTHOR]

8-Hydroxydaidzein (8-OHDe) is a non-natural isoflavone polyphenol isolated from fermented soybean foods. 8-OHDe exhibits a wide range of pharmaceutical activities. However, both the poor solubility and instability of 8-OHDe limit its applications. To resolve the limitations of 8-OHDe, Deinococcus geothermalis amylosucrase (DgAS) has previously been used to glycosylate 8-OHDe to produce soluble and stable 8-OHDe-7-O-α-glucopyranoside (8-OHDe-7-G) in a 0.5 h reaction time. In this study, we aimed to use DgAS and an extended reaction time to produce 8-OHDe diglucosides. At least three 8-OHDe derivatives were produced after a 24 h reaction time, and two major products were successfully purified and identified as new compounds: 8-OHDe-7-O-[α-glucopyranosyl-(1→6)-α-glucopyranoside] (8-OHDe-7-G2) and 8-OHDe-7,4′-O-α-diglucopyranoside (8-OHDe-7-G-4′-G). 8-OHDe-7-G-4′-G showed a 4619-fold greater aqueous solubility than 8-OHDe. In addition, over 92% of the 8-OHDe diglucosides were stable after 96 h, while only 10% of the 8-OHDe could be detected after being subjected to the same conditions. The two stable 8-OHDe diglucoside derivatives have the potential for pharmacological usage in the future. [ABSTRACT FROM AUTHOR]

This study evaluated the effects of iterative retrogradation on the structure and in vitro digestion of amylosucrase-treated waxy corn starch. Iterative retrogradation was conducted by repeating the set of autoclaving and retrogradation (4°C for 5 d) up to three times. After amylosucrase treatment, the average branch chain length increased. With retrogradation cycle count, melting enthalpy and temperature of amylosucrase-treated starch increased from 3.2 to 9.2 J/g and from 89.5°C to 95.2°C, respectively, and its relative crystallinity increased from 33.1% to 40.4%. By iterative retrogradation, the internal structures of amylosucrase-treated starch crystallites were altered to exhibit increased heat stabilities and perfections. In regards to in vitro digestibility, iteratively retrograded amylosucrase-treated starches showed higher RS content and lower RDS content compared with amylosucrase-treated starch. However, the dramatic changes in thermal transition properties, relatively crystallinity, and in vitro digestibility occurred during the first cycle of retrogradation and further retrogradation cycle did not affect them.

Extremophiles are nature's tiny warriors as they call inhospitable environments their home. They possess special factor(s) that offer an edge over other life forms susceptible to harsh conditions. One such family of extremophiles under discussion here is Deinococcaceae. The microbes belonging to Deinococcaceae are primarily radiophiles, the world's most radiation resistant bacteria, in addition to having resistance to high temperature, metals, cold etc. in specific species. Gamma rays have always been known to be lethal to living cells as it damages DNA, the blueprint of life. But, Deinococci sustain extremely high doses of gamma radiation, about 3000 times more than the dose humans succumb to. This review brings forth the utility of these special factors of Deinococcaceae for a broad range of biotechnological applications.

Faecalibacterium prausnitzii, a major commensal bacterium in the human gut, is well known for its anti-inflammatory effects, which improve host intestinal health. Although several studies have reported that inulin, a well-known prebiotic, increases the abundance of F. prausnitzii in the intestine, the mechanism underlying this effect remains unclear. In this study, we applied liquid chromatography tandem mass spectrometry (LC-MS/MS)-based multiomics approaches to identify biological and enzymatic mechanisms of F. prausnitzii involved in the selective digestion of inulin. First, to determine the preference for dietary carbohydrates, we compared the growth of F. prausnitzii in several carbon sources and observed selective growth in inulin. In addition, an LC-MS/MS-based intracellular proteomic and metabolic profiling was performed to determine the quantitative changes in specific proteins and metabolites of F. prausnitzii when grown on inulin. Interestingly, proteomic analysis revealed that the putative proteins involved in inulin-type fructan utilization by F. prausnitzii, particularly β-fructosidase and amylosucrase were upregulated in the presence of inulin. To investigate the function of these proteins, we overexpressed bfrA and ams, genes encoding β-fructosidase and amylosucrase, respectively, in Escherichia coli, and observed their ability to degrade fructan. In addition, the enzyme activity assay demonstrated that intracellular fructan hydrolases degrade the inulin-type fructans taken up by fructan ATP-binding cassette transporters. Furthermore, we showed that the fructose uptake activity of F. prausnitzii was enhanced by the fructose phosphotransferase system transporter when inulin was used as a carbon source. Intracellular metabolomic analysis indicated that F. prausnitzii could use fructose, the product of inulin-type fructan degradation, as an energy source for inulin utilization. Taken together, this study provided molecular insights regarding the metabolism of F. prauznitzii for inulin, which stimulates the growth and activity of the beneficial bacterium in the intestine.

Diverse flavonoid glycosides are present in the plant kingdom. Advanced technologies have been utilized to synthesize glycosyl flavonoids which exhibit good physicochemical characteristics. Previously, novel isoquercitrin (IQ) mono-, di-, and tri-glucosides (IQ-G1′, IQ-G2′, and IQ-G3′; atypical IQ-Gs (IQ-Gap)) were synthesized through the reaction of amylosucrase. Here, the regio-selective transglycosylation yields were predicted using response surface methodology for three variables (glucose donor (sucrose; 100–1500 mM), glucose acceptor (IQ; 100–400 µM), and pH (5.0–8.8)) using 1 unit/mL of enzyme at 45 °C; then, the optima were verified according to the experimental responses. Acidity (pH 5.0) was a major contributor for IQ-G1′ production (> 50%), and high sucrose concentration (1500 mM) limited IQ-G3′ production (< 15%). Low sucrose concentration (100 mM) at pH 7.0 promoted higher glycosyl IQ production (> 30%). Time-course production of IQ-Gap showed an exponential growth with different rates. IQ-Gap was stable under the simulated intestinal conditions compared with typical IQ-Gs. Digestive stable IQ-Gap can be effectively synthesized by modulating reaction conditions; thereby, atypical glycosyl products may contribute to the elucidation of nutraceutical potential of flavonoid glycosides. Key Points: •Predictions of RSM were validated for the regio-selective IQ-Gapproduction. • Time course changes of IQ-Gapindicate non-processive glycosylation of DGAS. • IQ-Gapexceed typical IQ-G in digestive stability at simulated intestinal condition. [ABSTRACT FROM AUTHOR]

Arbutin is a naturally occurring glycosylated product of hydroquinone. With the ability to interrupt melanin biosynthesis in epidermal cells, it is a promising cosmetic ingredient. In this study, a novel amylosucrase, Asmet, identified from a thermal spring metagenome, has been characterized for arbutin biosynthesis. Asmet was able to catalyze transglucosylation of hydroquinone to arbutin, taking sucrose as glycosyl donor, in the temperature range of 20 °C to 40 °C and pH 5.0 to 6.0, with the relative activity of 80% or more. The presence of chloride salts of Li, K, and Na at 1 mM concentration did not exhibit any notable effect on the enzyme's activity, unlike Cu, Ni, and Mn, which were observed to be detrimental. The hydroquinone (20 mM) to sucrose ratio of 1:1 to 1:10 was appropriate for the catalytic biosynthesis of arbutin. The maximum hydroquinone to arbutin conversion of 70% was obtained in 24 h of Asmet led catalysis, at 30 °C and pH 6.0. Arbutin production was also demonstrated using low-cost feedstock, table sugar, muscovado, and sweet sorghum stalk extract, as a replacement for sucrose. Whole-cell catalysis of hydroquinone to arbutin transglucosylation was also established. [ABSTRACT FROM AUTHOR]

The transglycosylation activity of Deinococcus geothermalis amylosucrase (DgAS) expressed in Corynebacterium glutamicum (cDgAS) was optimized for luteolin glycoside synthesis using purified enzymes {PEs), whole cells (WCs) suspended in buffer (WCB) and WCs in ethanol (WCE). Unlike PE, both WCB and WCE had high transglycosylation yields exceeding 95% over a wide temperature range, with cells in ethanol exhibiting the greatest enzymatic activity for up to 48 h due to membrane degradation and the slow release of cDgAS, which led to lysis and death. Meanwhile, in buffer, protection of the cytoplasmic membrane and ideal pH conditions maintained activity for over 200 h. To improve their performance, WCs were immobilized on agarose and the resulting immobilized cells (!Cs) had high stability in organic solvents, although WCB possessed greater thermal stability at 50 °c. The reaction parameters for PE, WCE, WCB, and IC transglycosylation were optimized using response surface methodology, and !Cs required lower concentrations of substrates while synthesizing higher concentrations of reaction products in addition to producing the novel compound luteolin maltotrioside after several cycles of reuse. This study expanded on the range of possibilities for the industrial application of C. glutamicum in increasing the production and bioavailability of novel and improved products. [ABSTRACT FROM AUTHOR]

Glycogen is a naturally occurring or metabolically synthesized biological macromolecule found in a wide range of living organisms, including animals, microorganisms, and even plants. However, naturally sourced glycogen poses challenges for industrial use. This study focused on a biological macromolecule referred to as glycogen-like particles (GLPs), detailing the production methods and biological properties of these particles. In vitro enzymatic production of GLPs was successfully achieved. GLPs synthesized through a simultaneous enzymatic reaction using sucrose had significant changes in their structure and functionality based on the branching enzyme (BE) to amylosucrase (ASase) ratio. As this ratio increased, the GLPs developed higher molecular weights and greater density, solubility, and branching degree while reducing size and turbidity. Structural changes in these enzymes were not observed beyond a critical BE/ASase ratio. Uniformly dispersed curcumin powder was generated in 50 % (w / v) aqueous GLP solution, and the GLPs were non-toxic to human skin keratinocytes at a concentration of 2.5 mg/mL. GLPs with lower branching inhibited tyrosinase activity and melanin synthesis, while those with more long chains displayed effective UV-blocking. By manipulating the BE/ASase ratio, GLPs were shown to display diverse chemical structures and physical characteristics, suggesting their potential application in the food and cosmetics industries. • GLPs are synthetic particles that are made from sucrose and enzymes. • The properties of GLPs can be tuned by varying the BE/ASase ratio. • GLPs help a uniform dispersion of curcumin. • GLPs with higher long chains give effective UV protection. • GLPs have potential applications in the food and cosmetic industries. [ABSTRACT FROM AUTHOR]

Abstract α-Arbutin is an effective skin-whitening cosmetic ingredient and hyperpigmentation therapy agent. It can be synthesized by one-step enzymatic glycosylation of hydroquinone (HQ), but limited by the low yield. Amylosucrase (Amy-1) from Xanthomonas campestris pv. campestris 8004 was recently identified with high HQ glycosylation activity. In this study, whole-cell transformation by Amy-1 was optimized and process scale-up was evaluated in 5000-L reactor. In comparison with purified Amy-1, whole-cell catalyst of recombinant E. coli displays better tolerance against inhibitors (oxidized products of HQ) and requires lower molar ratio of sucrose and HQ to reach high conversion rate (> 99%). Excess accumulation of glucose (0.6–1.0 M) derived from sucrose hydrolysis inhibits HQ glycosylation rate by 46–60%, which suggests the importance of balancing HQ glycosylation rate and sucrose hydrolysis rate by adjusting the activity of whole-cell catalyst and HQ-fed rate. Using optimal conditions, 540 mM of final concentration and 95% of molar conversion rate were obtained within 13–18 h in laboratory scale. For industrial scale-up production, 398 mM and 375 mM of final concentration with high conversion rates (~ 95%) were obtained in 3500-L and 4000-L of reaction volume, respectively. These yields and productivities (4.5–4.9 kg kL−1 h−1) were the highest by comparing to the best we known. Hence, high-yield production of α-arbutin by batch-feeding whole-cell biotransformation was successfully achieved in the 5000-L reaction scale.

Puerarin (daidzein-8-C-glucoside) is an isoflavone isolated from several leguminous plants of the genus Pueraria. Puerarin possesses several pharmacological properties; however, the poor solubility of puerarin limits its applications. To resolve this poor solubility, Deinococcus geothermalis amylosucrase (DgAS) was used to modify puerarin into more soluble derivatives. The results showed that DgAS could biotransform puerarin into a novel compound: puerarin-4′-O-α-glucoside. The biotransformation reaction was manipulated at different temperatures, pH values, sucrose concentrations, reaction times, and enzyme concentrations. The results showed that the optimal reaction condition was biotransformed by 200 μg/mL DgAS with 20% (w/v) sucrose at pH 6 and incubated at 40 °C for 48 h, and the optimal production yield was 35.1%. Puerarin-4′-O-α-glucoside showed 129-fold higher solubility than that of puerarin and, thus, could be further applied for pharmacological use in the future.

Glycosylation occurring at either lipids, proteins, or sugars plays important roles in many biological systems. In nature, enzymatic glycosylation is the formation of a glycosidic bond between the anomeric carbon of the donor sugar and the functional group of the sugar acceptor. This study found novel glycoside anomers without an anomeric carbon linkage of the sugar donor. A glycoside hydrolase (GH) enzyme, amylosucrase from Deinococcus geothermalis (DgAS), was evaluated to glycosylate ganoderic acid F (GAF), a lanostane triterpenoid from medicinal fungus Ganoderma lucidum, at different pH levels. The results showed that GAF was glycosylated by DgAS at acidic conditions pH 5 and pH 6, whereas the activity dramatically decreased to be undetectable at pH 7 or pH 8. The biotransformation product was purified by preparative high-performance liquid chromatography and identified as unusual α-glucosyl-(2→26)-GAF and β-glucosyl-(2→26)-GAF anomers by mass and nucleic magnetic resonance (NMR) spectroscopy. We further used DgAS to catalyze another six triterpenoids. Under the acidic conditions, two of six compounds, ganoderic acid A (GAA) and ganoderic acid G (GAG), could be converted to α–glucosyl-(2→26)-GAA and β–glucosyl-(2→26)-GAA anomers and α-glucosyl-(2→26)-GAG and β-glucosyl-(2→26)-GAG anomers, respectively. The glycosylation of triterpenoid aglycones was first confirmed to be converted via a GH enzyme, DgAS. The novel enzymatic glycosylation-formed glycoside anomers opens a new bioreaction in the pharmaceutical industry and in the biotechnology sector.

Amylosucrase (ASase, E.C. 2.4.1.4) is a powerful transglycosylation enzyme that can transfer glucose from sucrose to the hydroxyl (-OH) group of various compounds. In this study, recombinant ASases from Deinococcus geothermalis (DgAS) and Bifidobacterium thermophilum (BtAS) were used to synthesize biosurfactants based on the computational analysis of predicted docking simulations. Successful predictions of the binding affinities, conformations, and three-dimensional structures of three surfactants were computed from receptor-ligand binding modes. DgAS and BtAS were effective in the synthesis of biosurfactants from glyceryl caprylate, glyceryl caprate, and polyglyceryl-2 caprate. The results of the transglycosylation reaction were consistent for both ASases, with glyceryl caprylate acceptor showing the highest concentration, as confirmed by thin layer chromatography. Furthermore, the transglycosylation reactions of DgAS were more effective than those of BtAS. Among the three substrates, glyceryl caprylate glycoside and glyceryl caprate glycoside were successfully purified by liquid chromatography–mass spectrometry (LC–MS) with the corresponding molecular weights. [ABSTRACT FROM AUTHOR]

The prevalence of inflammatory bowel disease (IBD) is increasing worldwide, and the key risk factor is high sugar diet. The objective of this study was to investigate anti-inflammatory effects of turanose on IBD in a dextran sulfate sodium (DSS)-induced colitis mouse model. An amylosucrase from Neisseria sicca (NsiAS) was newly identified and successfully expressed. This enzyme exhibited optimal activity at 45 °C and pH 8.5. By utilizing this recombinant enzyme expressed in the E. coli host, turanose yield maximally reached 47% from 2.0 M sucrose. Colitis was induced with two 5-d cycles of 2.5% DSS in drinking water with a 10-d inter-cycle interval. Mice were fed AIN93G diet with 25% and 50% turanose replacement. Improved disease activity index scores, colon length, histopathological features, and myeloperoxidase activity were observed in the turanose supplemented group. Meanwhile, decreases in micro RNA (miR)-21 expression, histone acetylation, expression of pro-inflammatory cytokines, and phosphorylated ERK and STAT3 were observed in colon tissues. Notably, miR-21 and histone acetylation levels were found to positively correlated with expression of tumor necrosis factor α. In conclusion, turanose can attenuate colitis via regulatory effects on miRNA-21 expression and histone acetylation-related proinflammatory mediators. These findings also support further exploration of turanose as a potential therapeutic sugar substitute for patients with IBD.

Amylosucrase (ASase) is α-glucan-producing enzyme. Four putative ASase genes (bdas , blas , bpas , and btas) were cloned from Bifidobacterium sp. and expressed in Escherichia coli. All ASases from Bifidobacterium sp. (BAS) displayed typical ASase properties with slightly different characteristics. Among the BASs studied, BdAS and BpAS showed maximal enzyme activities at 35 and 30 °C, respectively, whereas BlAS and BtAS were maximally active at higher temperatures, i.e., 45 and 50 °C, respectively. BpAS exhibited optimum pH under slightly basic conditions (pH 8.0), while BdAS, BlAS, and BtAS preferred weakly acidic conditions (pH 5.0–6.0). All BASs showed higher isomerization activities. Particularly, BlAS produced more trehalulose than turanose. Although polymerization was the highest for BtAS, BtAS synthesized α-1, 4-glucans with a lower degree of polymerization than that of the other BASs. The versatile properties of the BASs described could contribute to the efficient production of highly valuable biomaterials for the agriculture, food, and pharmaceutical industries. • Four amylosucrases from Bifidobacterium (BdAS, BlAS, BpAS, and BtAS) were studied. • BlAS and BpAS have lower substrate affinities for sucrose than BdAS and BtAS. • The lower polymerization of BdAS may link to a six critical residues (HDDIGW). • Trehalulose production of BlAS was superior among four amylosucrases. • The study on BASs contributes to the efficient application of valuable biomaterials. [ABSTRACT FROM AUTHOR]

Modification of chestnut starch with amylosucrase from Deinococcus geothermalis (DGAS) increases the proportion of resistant starch. DGAS-modified chestnut starch (DMCS) attenuates obesity in diet-induced obese mice via a receptor of short chain fatty acids (SCFAs), G-protein-coupled receptor 43. SCFAs are gut microbial metabolites produced by fermenting resistant starch and have key roles in the obesity-ameliorating effects of DMCS. Here, we evaluated the mechanical links among DMCS-induced changes in the gut microbiota, consequent production of microbial metabolites, and host genetic responses. Supplementation with DMCS altered the proportions of cecal microbiota, such as Ruminococcaceae and Bacteroides ; microbial metabolites, such as acetic acid; and some carbohydrate metabolites. DMCS also induced changes in the expression of some genes in cecal epithelial cells, including genes involved in energy production, the cell cycle, and cellular junctions. Changes in the gut microbiota, microbial metabolites, and host gene expression were found to be significantly correlated. Our findings demonstrated the integrated and incorporated association among the gut microbiota, their beneficial metabolites, and the host transcriptome, which contributed to clarifying the anti-obesity effects of DMCS as a prebiotic. Therefore, fortifying resistant starch by modification of chestnut starch using DGAS may be a good strategy in the functional food industry. • Acetic acid as a mediator of the health-benefit was produced by gut microbiota by fermenting resistant starch in DMCS. • Identification of the prebiotic characteristics of DMCS through multi-omics analyses. • Increased expression of genes related to cell cycle and cell components in DMCS-supplemented mice. • The activation of energy production mechanism by gut microbial metabolites acetic acid in DMCS-supplemented mice. [ABSTRACT FROM AUTHOR]

Amylosucrase (AS; EC 2.4.1.4) is an enzyme that has great potential in the biotechnology and food industries, due to its multifunctional enzyme activities. It can synthesize α-1,4-glucans, like amylose, from sucrose as a sole substrate, but importantly, it can also utilize various other molecules as acceptors. In addition, AS produces sucrose isomers such as turanose and trehalulose. It also efficiently synthesizes modified starch with increased ratios of slow digestive starch and resistant starch, and glucosylated functional compounds with increased water solubility and stability. Furthermore, AS produces turnaose more efficiently than other carbohydrate-active enzymes. Amylose synthesized by AS forms microparticles and these can be utilized as biocompatible materials with various bio-applications, including drug delivery, chromatography, and bioanalytical sciences. This review not only compares the gene and enzyme characteristics of microbial AS, studied to date, but also focuses on the applications of AS in the biotechnology and food industries. [ABSTRACT FROM AUTHOR]

Amylosucrase (EC 2.4.1.4) is a versatile enzyme with significant potential in biotechnology and food production. To facilitate its efficient preparation, a novel expression strategy was implemented in Bacillus licheniformis for the secretory expression of Neisseria polysaccharea amylosucrase (NpAS). The host strain B. licheniformis CBBD302 underwent genetic modification through the deletion of sacB, a gene responsible for encoding levansucrase that synthesizes extracellular levan from sucrose, resulting in a levan-deficient strain, B. licheniformis CBBD302B. Neisseria polysaccharea amylosucrase was successfully expressed in B. licheniformis CBBD302B using the highly efficient Sec-type signal peptide SamyL, but its extracellular translocation was unsuccessful. Consequently, the expression of NpAS via the twin-arginine translocation (TAT) pathway was investigated using the signal peptide SglmU. The study revealed that NpAS could be effectively translocated extracellularly through the TAT pathway, with the signal peptide SglmU facilitating the process. Remarkably, 62.81% of the total expressed activity was detected in the medium. This study marks the first successful secretory expression of NpAS in Bacillus species host cells, establishing a foundation for its future efficient production., One-Sentence Summary: Amylosucrase was secreted in Bacillus licheniformis via the twin-arginine translocation pathway., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Enzymatically modified starch with controllable digestibility may find unique applications. In the present study, side chains of amylopectin were elongated by amylosucrase, followed by debranching with pullulanase to produce linear chains that self-assembled to form microparticles containing aggregated nanoparticles. Amylosucrase treatment for a longer time resulted in longer branches that were not changed after the subsequent modification by pullulanase. The A-type crystalline structure of native amylopectin was changed to the B-type and the percentage of crystallinity progressively increased after amylosucrase and subsequent pullulanase treatments. Elongation of branch chains favored the formation of thermal stable double helices, which decreased the contents of slowly digestible and rapidly digestible starches and increased the content of resistant starch from 9.1% of native amylopectin to be as much as 67.2% of microparticles. This study may provide an efficient and environmentally benign technology to produce modified starch with controllable physical and digestion properties enabling novel applications. Image 1 • Side chains of amylopectin were progressively elongated by amylosucrase. • Branching points of modified amylopectin were hydrolyzed by pullulanase. • Microparticles produced from linear chains containing aggregated nanoparticles. • Digestibility of amylopectin after sequential enzymatic treatments were evaluated. [ABSTRACT FROM AUTHOR]

Amylosucrase (AS) is a glycosyltransferase that produces linear α-1,4 glucans using sucrose as the sole substrate. In this study, for various applications, α-glucan-coated starch (α-GCS) was produced by AS (20 U/L reactant) from Neisseria polysaccharea to improve the physicochemical properties of raw normal corn starch (NCS) by applying different reaction conditions (i.e., varying the substrate concentration, pH, and temperature). Field-emission scanning electron microscopy (FE-SEM) and X-ray diffraction (XRD) showed that raw NCS was successfully coated by α-glucan. Differential scanning calorimetry (DSC) and rapid viscosity analyses (RVA) of the α-GCS confirmed that the α-glucan coating decreased the degree of retrogradation. Notably, compared to raw NCS as a control, starch retrogradation was significantly (p < 0.05) decreased by 13.7% after five weeks. Therefore, the novel α-GCS can be applied as a functional material for controlled retrogradation in the starch-based food industry for shelf-life extension. • α-Glucan-coated starch (α-GCS) was produced from raw starch by amylosucrase. • α-GCS retained the inherent physicochemical properties of raw starch. • The retrogradation level of α-GCS decreased compared to that of raw starch. • α-GCS can be applied as a functional food ingredient with extended shelf-life. [ABSTRACT FROM AUTHOR]

In this study, we improved the polymerization activity of Deinococcus geothermalis amylosucrase (DGAS) via semi-rational design to support increased potential industrial applications. Structural analysis selected three amino acid residues (Pro219, Phe225, and Ala226) in loop 3 of the B domain that were considered to be related to polymerization activity. Various DGAS variants were constructed through site-saturated mutagenesis, from which two mutants (DGAS-P219Y and DGAS-A226N) with increased polymerization activity were selected by fluorescence screening. DGAS-A226N synthesized longer amylose-like polymers from sucrose than those from wild-type (WT) DGAS. A kinetic study of sucrose as a substrate showed that the ratio of polymerization to hydrolysis activity of DGAS-A226N was significantly higher than that of WT DGAS. DGAS-A226N exhibited its highest enzymatic activity at 50 °C; its half-life was 6.8 min at 60 °C, and its melting temperature was 61.16 ± 0.09 °C. Computational simulation analysis of DGAS-A226N indicated that its conformation was more compact than that of WT DGAS, possibly owing to decreased flexibility of loops 2, 3, 4, 7, and 8. Thus, conformational changes in DGAS-A226N allowed the production of longer oligosaccharides, which may support a broader range of industrial application by affording novel or improved product characteristics. • An improved DGAS polymerization activity variant was made via semi-rational design. • Two mutants (P219Y and A226N) were selected by fluorescence-based screening. • DGAS-A226N synthesized longer amylose-like polymers from sucrose than WT DGAS. • DGAS-A226N loop 2, 3, 4, 7, and 8 flexibilities were decreased compared to WT DGAS. • DGAS-A226N conformational changes increased polymerization activity from WT enzyme. [ABSTRACT FROM AUTHOR]

Abstract Enzymatically-modified resistant starch (RS) was prepared by amylosucrase (ASase) reaction and was applied to develop structurally strengthened rice starch gels. Effects of ASase-modified and commercial RSs were examined for textural and physicochemical properties of rice starch gels. Rice gel hardness increased up to 60% with the presence of ASase-modified RS; the increase was proportional to the substitution level. Gel cohesiveness seemed to decline significantly, while springiness kept intact when RSs were added. A slight decrease in elasticity was observed for the sample with 20% (w/w) of ASase-modified normal corn starch, while 2–5 times higher RS content in this sample was obtained compared to untreated gel. Rice starch gels reinforced with ASase-modified RS decreased up to 40% of water exudation from gel structure, and intact morphological structure was observed after 7-day storage. These results suggested that ASase-modified RS would be applicable for producing rice starch gel foods as a texture enhancer and a functional ingredient. Graphical abstract Image 1 Highlights • Firmness of rice starch gel was improved by enzymatically-made resistant starch (RS). • This RS supplementation greatly increased dietary fiber content in rice starch gels. • Rice gel structure by supplementing RS was greatly stabilized with less syneresis. • Rice gel elasticity was not negatively affected by replacing up to 20% of the RS. [ABSTRACT FROM AUTHOR]

In this study, starch–lipid complexes were prepared using normal corn starch (NC) and amylosucrase‐modified waxy corn starch (ASWC) with myristic acid (C14:0) and palmitic acid (C16:0). The amylosucrase modification elongated branch chains in waxy corn starch leading to an increase of apparent amylose content (29.7%) similar to that of NC (29.0%). The X‐ray diffraction of starch–lipid complexes revealed a V‐type pattern, a clear indication of complex formation. The ability of the ASWC to complex with fatty acids was greater than that of NC. Interestingly, the changes in relative crystallinity, thermal parameters, and digestion properties according to the complexation showed opposite patterns in NC and ASWC. This study found that the structure of ASWC contributes to the formation of starch–fatty acid complexes and suggested that the ASWC can be preferred over NC in a delivery system. Practical Application: Amylopectin has been considered to be incapable of forming complexes with fatty acids due to its short chain length and steric hindrance. Through this study, an appropriate enzymatic modification of the molecular structures of waxy starches could make a complexation of waxy starches with fatty acids possible. The findings of this study suggest a promising perspective for utilization of waxy starch as a carrier material of lipophilic molecules. [ABSTRACT FROM AUTHOR]

α-Arbutin is an effective skin-whitening cosmetic ingredient and can be synthesized through hydroquinone glycosylation. In this study, amylosucrase (Amy-1) from Xanthomonas campestris pv. campestris 8004 was newly identified as a sucrose-utilizing glycosylating hydroquinone enzyme. Its kinetic parameters showed a seven-time higher affinity to hydroquinone than maltose-utilizing α-glycosidase. The glycosylation of HQ can be quickly achieved with over 99% conversion when a high molar ratio of glycoside donor to acceptor (80:1) was used. A batch-feeding catalysis method was designed to eliminate HQ inhibition with high productivity (> 36.4 mM h−1). Besides, to eliminate the serious inhibition caused by the accumulated hydroquinone oxidation products, the whole-cell catalysis was further proposed. 306 mM of α-arbutin was finally achieved with 95% molar conversion rate within 15 h. Hence, the batch-feeding whole-cell biocatalysis by Amy-1 is a promising technology for α-arbutin production with enhanced yield and molar conversion rate. [ABSTRACT FROM AUTHOR]

Highlights • A new thermostable amylosucrase was isolated from Bifidobacterium thermophilum (Bt AS). • Sucrose biomass was converted biocatalytically to low-calorie functional turanose at 50 °C. • Molecular size of linear α-glucan produced by Bt AS was not affected by [sucrose]. Abstract A unique thermostable amylosucrase from Bifidobacterium thermophilum was produced as a recombinant protein with the half-life of 577 h at 50 °C. By adding 1.0 M fructose, turanose yield was improved from 22.7% to 43.3% with 1.0 M sucrose, and from 23.7% to 39.4% with 1.5 M sucrose. Sucrose consumption rate was greatest at 55 °C, but the lowest amount of turanose was produced. Thus, turanose yield from sucrose biomass was inversely proportional to reaction temperature and was highly dependent on [fructose]. Meanwhile, insoluble α-glucan yield was clearly reduced as [fructose] increased. With 1.0 M fructose + 1.0 M sucrose, glucan byproduct yield significantly decreased from 29.4% to 1.1%. Molecular weights of linear glucans were almost identical among various [sucrose]s and were homogenous with very low polydispersity. This unique dual reaction patterns of amylosucrase enzyme would be very useful for massive productions of two different biomaterials simply by changing sucrose biomass concentration. [ABSTRACT FROM AUTHOR]

Amylosucrases (ASase, EC 2.4.1.4) from Deinococcus geothermalis (DGAS) and Neisseria polysaccharea (NPAS) were heterologously expressed in Bacillus subtilis. While DGAS was successfully expressed, NPAS was not. Instead, NPAS was expressed in Escherichia coli. Recombinant DGAS and NPAS were purified using nickel-charged affinity chromatography and employed to modify daidzin to enhance its water solubility and bioavailability. Analyses by LC/MS revealed that the major products of transglycosylation using DGAS were daidzein diglucoside and daidzein triglucoside, whereas that obtained by NPAS was only daidzein diglucoside. The optimal bioconversion conditions for daidzein triglucoside, which was predicted to have the highest water-solubility among the daidzin derivatives, was determined to be 4% (w/v) sucrose and 250 mg/L daidzin in sodium phosphate pH 7.0, with a reaction time of 12 h. Taken together, we suggest that the yield and product specificity of isoflavone daidzin transglycosylation may be modulated by the source of ASase and reaction conditions. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Amylosucrase and CGTases were compared to generate α -glycosyl isoquercitrin. • Amylosucrase has several pros over CGTase to generate α -glycosyl isoquercitrin. • Amylosucrase showed bioconversion yield up to 97%. • Amylosucrase produced quercetin-3- O -tetra-glucoside over 5 folds higher than CGTase. Abstract Isoquercitrin (IQ, quercetin-3- O - β - d -glucopyranoside) has diverse biological functions, such as anti-oxidant and anti-cancer activity, but its use is limited by poor solubility and bioavailability. Enzymatically modified IQ (EMIQ) is a mixture of transglycosylated IQs that have better solubility and bioavailability than do quercetin and IQ. Two different enzymes, cyclodextrin glucanotransferase (CGTase) and amylosucrase (ASase), have the transglycosylation activity to produce EMIQ. Both enzymes produce a variety of EMIQs including IQ, IQ-glucoside (IQ-G1), IQ-diglucoside (IQ-G2), and IQ-triglucoside (IQ-G3). ASase had a higher bioconversion yield from IQ to EMIQ (97.6%) than did CGTase (76.8%). In addition, the yield of IQ-G3, which was the most bioavailable form, was higher with ASase (46%) than with CGTases (8%). Taken together, these results suggest that ASase can be used to synthesize EMIQ in a simple and specific process. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A novel α-amylase from Pseudomonas mendocina was partially characterized. • A dual enzyme strategy was developed to produce MOS from sucrose. • In the one-pot enzymatic reaction, a maximum MOS yield of 22.8% was achieved. • In the two-step enzymatic reaction, the highest MOS production yield was 34.1%. Abstract Maltooligosaccharides (MOS), oligosaccharides composed of 2 to 10 glucopyranose subunits linked by α-1,4 bonds, are promising ingredients possessing low sweetness, low osmolality, high water-holding capacity, and crystallization-prevention ability. For improved the cost effectiveness, a novel process for MOS preparation was developed by utilizing sucrose as a raw material instead of starch. In this work, a novel α-amylase from Pseudomonas mendocina (PM-Amy) was characterized and applied to produce the MOS from α-1,4-glucans obtained by the amylosucrase treatment of sucrose. PM-Amy showed a maximum activity at a pH of 6.0 and 55 °C. In the one-pot enzymatic reaction, a maximum MOS yield of 22.8% (w/w) was achieved, while the highest MOS yield (34.1%, w/w) was achieved from the two-step dual enzyme reaction. [ABSTRACT FROM AUTHOR]